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ABSTRACT In this work, we collected foodborne Salmonella strains in Shenzhen, China, during 2014–2017 and investigated the genetic profile of all cefotaxime-resistant isolates in the collection. The strains were subjected to antimicrobial susceptibility tests, whole-genome sequencing, bioinformatics analysis, and conjugation studies. A total of 79 cefotaxime-resistant Salmonella were identified and found to exhibit multidrug resistance. Resistance rate recorded during the study period increased from 1.9% to 9.1%. Salmonella Typhimurium was the predominant serovar, and CTX-M family genes were dominant among the ESBLs genes detected. Notably, CTX-M-bearing plasmids or transposons often contain other drug resistance genes. Furthermore, a combination of CTX-M-55 and CTX-M-65 genes was detected for the first time in foodborne Salmonella strains. Our findings reveal the prevalence and molecular characteristics of cefotaxime-resistant foodborne Salmonella strains in southern China. IMPORTANCE Cefotaxime-resistant Salmonella strains pose an increasing threat to human health by causing infections with limited treatment options. It is therefore necessary to undertake a surveillance on the prevalence of such strains and investigate the resistance and transmission mechanisms. In this work, various ESBL genes flanked by different IS located in different mobile genetic elements were detectable among cefotaxime-resistant Salmonella strains. These data show that the high prevalence and genotypic diversity of cefotaxime-resistant foodborne Salmonella strains in China are possibly attributed to the evolution and transmission of a wide range of multidrug resistance-encoding mobile genetic elements.

ABSTRACT Plasmid-mediated quinolone resistance (PMQR) determinants, such as qnrVC genes, have been widely reported in Vibrio spp. while other types of PMQR genes were rarely reported in these bacteria. This study characterized the phenotypic and genotypic features of foodborne Vibrio spp. carrying qnrS, a key PMQR gene in Enterobacteriaceae. Among a total of 1,811 foodborne Vibrio isolates tested, 34 (1.88%) were found to harbor the qnrS gene. The allele qnrS2 was the most prevalent, but coexistence with other qnr alleles was common. Missense mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes were only found in 11 of the 34 qnrS-bearing isolates. Antimicrobial susceptibility tests showed that all 34 qnrS-bearing isolates were resistant to ampicillin and that a high percentage also exhibited resistance to cefotaxime, ceftriaxone, and trimethoprim-sulfamethoxazole. Genetic analysis showed that these phenotypes were attributed to a diverse range of resistance elements that the qnrS-bearing isolates harbored. The qnrS2 gene could be found in both the chromosome and plasmids; the plasmid-borne qnrS2 genes could be found on both conjugative and nonconjugative plasmids. pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of phenotypic resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would speed up the emergence of multidrug-resistant (MDR) pathogens that are resistant to the most important antibiotics used in treatment of Vibrio infections, suggesting that close monitoring of emergence and dissemination of MDR Vibrio spp. in both food samples and clinical settings is necessary. IMPORTANCE Vibrio spp. used to be very susceptible to antibiotics. However, resistance to clinically important antibiotics, such as cephalosporins and fluoroquinolones, among clinically isolated Vibrio strains is increasingly common. In this study, we found that plasmid-mediated quinolone resistance (PMQR) genes, such as qnrS, that have not been previously reported in Vibrio spp. can now be detected in food isolates. The qnrS2 gene alone could mediate expression of ciprofloxacin resistance in Vibrio spp.; importantly, this gene could be found in both the chromosome and plasmids. The plasmids that harbor the qnrS2 gene could be both conjugative and nonconjugative, among which the pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would accelerate the emergence of multidrug-resistant pathogens.

ABSTRACT Expression of the hypermucoviscosity (HMV) phenotype and capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae were reported to be encoded by genes located in the chromosomal rmp locus. However, the functions of the rmp locus in the virulence plasmid remained unclear, and most of the rmp loci in clinical K. pneumoniae are plasmid carried. In this study, we investigated the functional characteristics of plasmid-borne rmp homologues in clinical hypervirulent K. pneumoniae (hvKP) strains by cloning and introducing such gene homologues into K. pneumoniae strains of different capsule types, followed by the evaluation of phenotypic changes in these strains. Acquisition of the plasmid-borne prmpADC and prmpA2D2 loci were found to result in an increase in mucoviscosity and CPS production in K1 and K2 K. pneumoniae, while only the prmpA2D2 locus contributed to phenotypic changes in the ST11/KL64 strain. Consistently, both rmpD and rmpD2 increased HMV in K1 and K2 K. pneumoniae, while only rmpD2 contributed to HMV in the ST11/KL64 strain; rmpC contributed to CPS overproduction in K1 and K2 strains but not in the ST11/KL64 strain. Furthermore, we proposed a logistic molecular basis of the HMV phenotype of K. pneumoniae on which prmpD2-mediated HMV is attributed to the increase of cell-free CPS production. Our data confirm that the rmp homologues carried by the virulence plasmid play a key role in virulence expression in K. pneumoniae, but the phenotype is highly dependent on the genetic background of the host strain and explained why most of the clinical ST11 strains carry only the prmpA2D2 locus. IMPORTANCE Klebsiella pneumoniae has become the most frequently isolated bacterial pathogen in hospital settings, with a very high mortality rate worldwide. Factors contributing to the virulence of K. pneumoniae are the overproduction of capsular polysaccharide (CPS) as well as the hypermucoviscosity (HMV) phenotype. These two phenotypes were reported to be regulated by rmpA/A2 homologues, which are often carried by virulence plasmids. Here, we determined the functional role of two plasmid-borne rmpA in mediating expression of the HMV phenotype and CPS production in K. pneumoniae. Different capsule types exhibited differences in the expression of HMV and CPS production although they harbored an identical plasmid-borne rmpA or rmpA2 locus, indicating that these virulence-related phenotypes are strongly related to the genetic background of the host strains. Our study provides a novel understanding of the regulation of virulence-related phenotypes and clinical management of K. pneumoniae infections.

ABSTRACT Mechanisms of azithromycin resistance have rarely been reported. In this study, an IncFIB/IncHI1B plasmid that confers resistance to azithromycin was recovered from a clinical Klebsiella pneumoniae strain. This plasmid could be efficiently disseminated to Escherichia coli, Salmonella, and other Gram-negative bacterial pathogens through conjugation. This plasmid was shown to carry three macrolide resistance genes: erm(B), a novel erm(42) gene, and mph(A). The functions of erm(42) were confirmed by direct cloning of this gene and determination of the MIC of azithromycin in strains of various bacterial species which have acquired this gene. Of particular concern is the potential transmission of azithromycin-resistance to extensively drug-resistant (XDR) Salmonella, which causes infections for which treatment options are extremely limited. Monitoring and preventing dissemination of this azithromycin resistance-encoding conjugative plasmid in Enterobacteriaceae is of utmost importance. IMPORTANCE In this study, we identified a conjugative plasmid carrying a novel azithromycin resistance gene, erm(42), from a clinical K. pneumoniae strain. Conjugation of this plasmid into Salmonella conjugants conferred resistance to azithromycin, which is considered a choice for treating Salmonella infections. Of particular concern is the dissemination of this type of azithromycin resistance-encoding conjugative plasmid to extensively drug-resistant (XDR) Salmonella. The study shows that further monitoring of the dissemination of this plasmid in clinical strains of Salmonella spp. is warranted.

ABSTRACT Colistin is a last-line antibiotic which acts by causing membrane permeabilization in Gram-negative bacteria. However, its clinical value has been limited by its toxicity and the emergence of resistant organisms. In this study, we showed that econazole and colistin can act synergistically to produce a strong antimicrobial effect sufficient for eradication of starvation-induced tolerant and multidrug-resistant populations of Acinetobacter baumannii, a notorious pathogen causing recalcitrant infections, both in vitro and in mouse infection models. Investigation of the underlying mechanism showed that, while colistin disrupts the membrane structure, econazole causes the dissipation of proton motive force, eliciting a vicious cycle of membrane structural damages and disruption of membrane protein functions, and eventually cell death. This drug combination therefore achieves our goal of using a much smaller dosage of colistin to produce a much stronger antimicrobial effect to tackle the problems of toxicity and resistance associated with colistin usage. IMPORTANCE Findings described in this study constitute concrete evidence that it is possible to significantly enhance the antimicrobial activity of colistin by using an antifungal drug, econazole, as a colistin adjuvant. We showed that this drug combination can kill not only multidrug-resistant A. baumannii but also the tolerant subpopulation of such strains known as persisters, which may cause chronic and recurrent infections in clinical settings. The synergistic killing effect of the econazole and colistin combination was also observable in mouse infection models at a very low concentration, suggesting that such a drug combination has high potential to be used clinically. Findings in this study therefore have important implications for enhancing its clinical application potential as well as developing new approaches to enhance treatment effectiveness and reduce suffering in patients.

ABSTRACT Coexistence of oqxAB and aac(6′)-Ib-cr is often associated with the expression of fluoroquinolone resistance in Salmonella. The actual role of the plasmid-borne oqxAB gene and its regulatory mechanism compared to its chromosomally encoded counterpart in Klebsiella pneumoniae remain unclear We found that cloning of oqxAB gene only or chromosomally encoded oqxABR (ABRc) locus did not lead to an increase of ciprofloxacin (CIP) minimum inhibitory concentration (MIC) in S. Typhimurium, while cloning of the plasmid-encoded oqxABR (ABRp) locus led to a 4-fold increase in CIP MIC, reaching 0.0065 μg/mL. The co-carriage of these constructs with aac(6′)-Ib-cr further increased the CIP MIC to 0.25 μg/mL in S. Typhimurium carrying aac(6′)-Ib-cr and ABRp. Analysis of the transcription start site sequences showed that the expression level of suppressor protein gene, oqxR, in strains carrying ABRp was lower than that of its chromosomal counterpart due to the truncated promoter region in ABRp. The lower expression of OqxR in ABRp led to the overexpression of OqxAB, which elevated CIP MIC and exhibited a synergistic antimicrobial effect with the aac(6′)-Ib-cr gene product to confer intermediate CIP (MIC = 0.25 μg/mL) in S. Typhimurium. Global transcriptional regulators in S. Typhimurium did not seem to play a role in regulating the plasmid-borne oqxAB genes. In conclusion, findings in this work showed that neither aac(6′)-Ib-cr nor oqxABRp, but the combination of both genes, could mediate intermediated resistance to fluoroquinolone in Salmonella. The truncated promoter region in the oqxR gene of the plasmid-encoded locus led to the constituted expression of oqxAB genes. IMPORTANCE The transferable mechanisms of quinolone resistance (TMQR) gene, oqxAB, has been widely detected in Salmonella and is commonly associated with aac(6′)-Ib-cr. It is thought to be associated with fluoroquinolone resistance, while its ancestor gene from K. pneumoniae is not. This study evaluated the actual role of the plasmid-borne oqxAB genes in Salmonella and showed that it was not able to mediate intermediated resistance to fluoroquinolone and only did so when it coexisted with aac(6′)-Ib-cr. Chromosomally encoded oqxABRc from K. pneumoniae was not able to mediate enhanced CIP MIC due to tight regulation by the suppressor oqxR. However, plasmid-encoded oqxABRp enabled oqxAB to be expressed constitutionally due to the truncated promoter region of oqxR, leading to lower expression of the suppressor oqxR. This study clarified the roles of oqxAB and aac(6′)-Ib-cr in mediating fluoroquinolone resistance in Salmonella and provides insights into the regulation of plasmid-encoded TMQR determinant, oqxAB.

ABSTRACT In this study, an IncI1 plasmid encoding resistance to both cefotaxime and azithromycin was recovered from a clinical Klebsiella pneumoniae strain. The azithromycin resistance was confirmed to be mediated by the erm(B) gene. This plasmid could be readily conjugated to strains of Escherichia coli and Salmonella, promoting rapid dissemination of azithromycin- and ceftriaxone-resistance-encoding elements among Gram-negative bacterial pathogens. Transmission of this plasmid in Salmonella is of particular concern, since it could mediate expression of phenotypic resistance to azithromycin and ceftriaxone, which are the current choices for treatment of Salmonella infections. Our findings suggest a need to monitor the efficiency and pattern of transmission of this plasmid among key Gram-negative bacterial pathogens. IMPORTANCE Since the approval by the FDA of azithromycin for treatment of Salmonella infections, efforts have been made to monitor the development of resistance to azithromycin in these organisms. In this study, we report an IncI1 plasmid from a clinical K. pneumoniae strain that encodes resistance to both cefotaxime and azithromycin. This plasmid could be readily conjugated to strains of Escherichia coli and Salmonella, promoting rapid dissemination of azithromycin- and ceftriaxone-resistance-encoding elements among Gram-negative bacterial pathogens. Furthermore, data from this study confirmed for the first time the role of the erm(B) gene in mediating resistance to azithromycin in various bacterial species, particularly Salmonella.

ABSTRACT Our objective was to characterize the genetic features of plasmids harbored by two genetically related, MCR-1 and NDM-5-producing Escherichia coli strains recovered from a chicken meat sample. The genetic profiles of all plasmids harbored by the two test strains, namely, 1106 and 1107, were determined by whole-genome sequencing, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and bioinformatics analysis. The transferability of plasmids harbored by the two strains was assessed by filter mating assay. Strains 1106 and 1107 were resistant to almost all the antibiotics, including colistin and fosfomycin, but remained susceptible to amikacin and tigecycline. The plasmids of p1107-NDM-5 and p1106-NDM-5 both contain a class I integron which lacks the ISAba125 element. The backbone of p1106-IncFII exhibited a high degree of similarity with that of p1106-NDM-5 and p1107-NDM-5, implying that events of plasmid fusion and resolution were involved in the formation of the two plasmids. The plasmids p1106-IncHI2MCR and p1107-IncHI2MCR belong to an IncHI2 replicon type, with three copies of ISApl1 being observed in p1106-IncHI2MCR, implying that the mcr-1 gene was transferable among bacteria that reside in the same food matrix. In this study, p1106-IncFIB, p1107-99K, p1107-111K, and p1107-118K were all found to be phage-like plasmids, with p1106-IncFIB and p1107-118K containing several virulence genes, including iroBCDEN, iucABCD, sitABCD, hlyF, and iss. Surprisingly, resistance genes such as aph(3′)-Ia, sul3, and aac(3′)-IId could also be found in p1107-118K, but resistance genes were not detected in other phage-like plasmids. In conclusion, enhanced surveillance is required to monitor and control the dissemination of various resistance determinants among foodborne pathogens. IMPORTANCE Carbapenem and colistin are last-resort antibiotics used to treat serious clinical infections caused by multidrug-resistant (MDR) bacterial pathogens. Plasmids encoding resistance to carbapenems and colistin have been reported in clinical pathogens in recent years, and yet few studies reported cocarriage of mcr and blaNDM genes in Escherichia coli strains of food origin. How plasmids encoding these two important resistance determinants are being evolved and transmitted in bacterial pathogens is not well understood. In this study, we investigated the genetic features of plasmids harbored by two nonclonal, mcr-1- and blaNDM-5-bearing E. coli strains (1106 and 1107) recovered from a fresh chicken meat sample to understand and provide evidence of the level and dynamics of MDR plasmid transmission. Our data confirmed that active plasmid fusion and resolution events were involved in the formation of plasmids that harbor multiple resistance genes, which provide insights into the further control of plasmid evolution in bacterial pathogens.

ABSTRACT Bacterial antibiotic tolerance, a phenomenon first observed in 1944, is known to be responsible for both onset and exacerbation of recurrent and chronic bacterial infections. The development of antibiotic tolerance was previously thought to be due to a switch to physiological dormancy when bacteria encounter adverse growth conditions. Our recent laboratory findings, however, showed that a set of genes related to the maintenance of proton motive force (PMF) are up-regulated under starvation, indicating that the tolerant sub-population, which are commonly known as persisters, can actively maintain their tolerance phenotypes. In this study, we investigated the relative functional roles of proteins involved in the maintenance and active generation of PMF in mediating tolerance formation in bacteria and found that the PspA and RcsB proteins play a key role in PMF maintenance in persisters, as deletion of genes encoding these two proteins resulted in significantly lower tolerance levels. Consistently, expression of the OsmC and Bdm proteins, which is under regulation by RcsB, is required to maintain PMF and the antibiotic tolerance phenotypes. On the other hand, the NuoL, Ndh, AppC, CyoB, and NuoF proteins, which are electron transport chain (ETC) components, were also found to be actively expressed in persisters in order to generate PMF to support functioning of various tolerance mechanisms such as efflux activities. Our data show that active generation of PMF is even more important than the PMF maintenance functions of PspA and RcsB in the expression of antibiotic tolerance phenotypes in persisters. Assessment of double- and triple-gene knockout strains, in which the PMF maintenance genes and those encoding ETC components were simultaneously deleted, confirms that these two groups of genes are both required for the expression of antibiotic tolerance phenotypes and that a lack of these functions would result in complete PMF dissipation and accumulation of antibiotics in the intracellular compartment of persisters and eventually cell death. Products of these genes are, therefore, ideal targets for future development of anti-tolerance agents. IMPORTANCE In this work, bacteria were found to undergo active generation and maintenance of proton motive force (PMF) under adverse conditions, such as starvation so as to support a range of physiological functions in order to survive under such conditions for a prolonged period. The ability to maintain a substantial level of PMF was found to be directly linked to that exhibiting phenotypic antibiotic tolerance under nutrient starvation or other adverse conditions. These findings infer that bacteria do not simply become physiologically dormant when they become antibiotic tolerant, instead they need to produce a wide range of proteins including those which help prevent PMF dissipation, such as PspA and RcsB, and the electron transport chain components, such as NuoL and Ndh, that actively generate PMF even during long-term starvation. As antibiotic tolerant sub-population is known to play a role in eliciting recurrent and chronic infections, especially among patients with a weakened immune system, the PMF maintenance mechanisms identified in this work are potential targets for the development of new strategies to control recurrent and chronic infections.

ABSTRACT Antibiotic tolerance is not only the key underlying the cause of recurrent and chronic bacterial infections but it is also a factor linked to exacerbation of diseases, such as tuberculosis, cystic fibrosis-associated lung infection, and candidiasis. This phenomenon was previously attributed to a switch to physiological dormancy in a bacterial subpopulation triggered by environmental signals. However, we recently showed that expression of phenotypic antibiotic tolerance during nutrient starvation is highly dependent on robust production of proteins that actively maintain the bacterial transmembrane proton motive force (PMF), even under a nutrient-deficient environment. To investigate why PMF needs to be maintained for expression of phenotypic antibiotic tolerance, we tested the relative functional role of known transporters and efflux pumps in tolerance development by assessing the effect of deletion of specific efflux pump and transporter-encoding genes on long-term maintenance of antibiotic tolerance in an Escherichia coli population under starvation. We identified eight specific efflux pumps and transporters and two known efflux pump components, namely, ChaA, EmrK, EmrY, SsuA, NhaA, GadC, YdjK, YphD, TolC, and ChaB, that play a key role in tolerance development and maintenance. In particular, deletion of each of the nhaA and chaB genes is sufficient to totally abolish the tolerance phenotypes during prolonged antimicrobial treatment. These findings therefore depict active, efflux-mediated bacterial tolerance mechanisms and facilitate design of intervention strategies to prevent and treat chronic and recurrent infections due to persistence of antibiotic-tolerant subpopulations in the human body. IMPORTANCE We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions. Intriguingly, we found that tolerance-maintaining efflux activities were mainly encoded by 10 efflux or transporter genes. Because our study showed that deletion of even one of these genes could cause a significant reduction in tolerance level, we conclude that the products of these genes play an essential role in enhancing the survival fitness of bacteria during starvation or under other adverse environmental conditions. These gene products are therefore excellent targets for future design of antimicrobial agents that eradicate antibiotic tolerant persisters and prevent occurrence of chronic and recurrent human infections.